The overall goal of this proposal is to gain a better understanding of how Ras proteins couple extracellular signals to intracellular signaling pathways in normal and disease states. The Ras superfamily of GTPases participate in the regulation of a wide variety of cellular processes by functioning as molecular switches that cycle between the active GTP-bound and inactive GTP-bound states. A key step in how Ras protein function is modulated in cells is by their activation by a family of guanine nucleotide exchange factors (GEFs), whose role is to respond to a specific extracellular signals by promoting the exchange of GTP for GDP bound to Ras proteins. This proposal will investigate how the Ras-GRF family of GEFs contribute to the control signaling pathways mediated by the Ras family of GTPases. Ras-GRF1 and Ras-GRF2 each have two GEF domains, one used to activate the Ras proto-oncogenes and another used to activate the Rac GTPase. Calcium/calmodulin binding regulates the ability of Ras-GRFs to activate Ras and Rac. Ras-GRFs can also be stimulated by phosphorylation. Both Ras-GRF proteins are expressed predominantly, but not exclusively in neurons. This proposal will use both genetic and biochemical approaches to focus on three major areas of study. The first is to reveal how the Ras-GRF family contributes to receptor/ calcium channel signaling in neurons by exploiting our recently derived mouse strains that lack Ras-GRF1, Ras-GRF2 or both Ras-GRFs. The second is to reveal how two classes of newly identified Ras-GRF binding proteins contribute to Ras GTPase regulation. These binding proteins include IB1 and IB2 (scaffolds for components of the Jnk and p38 MAP kinase cascades, respectively) and spinophilin (a scaffold for the p70 S6 kinase and protein phosphatase 1). Finally, the third goal is to investigate the biological function of the newly identified Ras-GRF1 target, R-Ras, in oncogenesis. These studies will exploit our recent finding that constitutively activated R-Ras is a potent activator of estrogen independent proliferation in MCF-7 breast cancer cell lines.